Int. J. Morphol., 25(1):73-83, 2007.

Cytological Aspects of the Differentiation of Barb Cells During the Formation of the Ramus of Feathers

Aspectos Otológicos de la Diferenciación de Células Barba Durante la Formación de las Ramas de las Plumas

Lorenzo Alibardi

Dipartimento di Biologia, University of Bologna, via Selmi 3, 40126 Bologna, Italy. Sponsorship. University of Bologna 605 Grant.

Correspondence to:

SUMMARY: The present ultrastructural study on developing and regenerating feathers of chick and zebrafinch describes the ultrastructural changes that occur during the differentiation of barb cells that leads to the formation of the ramus of barbs. Differently from barbule and barb cortical cells that accumulate feather keratin, barb medullary cells undergo to lipid degeneration. Eventually, lipids disappear and medullary cells become empty cavities in the central part of the ramus. In barb medullary cells feather keratin is accumulated in few peripheral bundles that merge with those of cortical cells to fom the wall of the ramus. The latter is joined with branching barbules. The process that controls the transition from keratin-synthesizing to lipid-producing barb cells remains unknown. The accumulation of lipids among keratin bundles confirms the capability of beta-keratin cells to undergo an intense lipidogenesis under specific conditions.

KEY WORDS: Feathers; Barbs; Differentiation; Lipidization; Ultrastructure.

RESUMEN: La presente investigación ultra estructural sobre el desarrollo y regeneración de plumas en polluelos y gorrión cebra (Taeniopygia guttata castanotis) describe los cambios ultraestructurales que pueden ocurrir durante la diferenciación de células barbas que lleva a la formación de las ramas de las barbas. Diferente a las barbas pequeñas y a las células barbas corticales que acumulan queratina en las plumas, las células barbas medulares se convierten en cavidades vacías en la parte central de la rama. En células barbas medulares la queratina de la pluma es acumulada en algunos fascículos periféricos que se unen con aquellos de las células corticales para formar la pared de la rama. Este último se une luego a pequeñas barbas en ramas. Aún es desconocido el proceso que controla la transición de la síntesis de queratina a células barbas produciendo lípidos. La acumulación de lípidos entre los acumulos de queratina confirma la capacidad de las células beta-queratina a someterse a una lípido génesis intensa bajo condiciones específicas.

PALABRAS CLAVE: Plumas; Barbas; Diferenciación; Lipidización; Ultraestructura.

INTRODUCTION

Feathers derive from a complex process of morphogenesis inside embryonic feather filaments that begins with the formation of barb ridges and terminates with the formation of barbs, free in plumulaceous feathers and regularly joined to arachis in pennaceous feathers (Lucas & Stettenheim, 1972; Chuong & Widelitz, 1999; Prum & Dyck, 2003; Sawyer & Knapp, 2003). Histologically, barbs are formed by an mid-axial part called ramus from which barbules branch laterally. Figs. 1 and 7 illustrate the general process of feather morphogenesis in order to show the process of formation of barbs. The mid-inner cell area of each barb ridge forms the ramus of the future barb while the two external and outer parts of the barb ridge, termed alar plates, form barbules (Figs. 1 and 2). The latter join to the centrally located ramus at different levels with a branching patterns that resemble plant ramifications (Fig. 1E; Alibardi, 2005a,b; Alibardi & Sawyer, 2006). Barb cells pile-up to form the ramus that eventually form a medullated part where large air cavities are present surrounded by a cortical part made of resistant feather keratin that sustain the whole barb.

The formation of the extended cavities in the medulla of rami derives from the degeneration of the central group of barb cells (medullary) that instead of accumulate the resistant feather keratin like the other barb cells (cortical) and barbule cells undergo a process of vacuolization (Matulionis, 1970; Alibardi, 2005a). Despite numerous studies on feather structure, the cytological details of the differentiation of barb medullary cells have been briefly described only in developing downfeathers of the chick (Matulionis; Alibardi, 2005a). The present study aims to deepen the ultrastructural description of the cell process of ramus differentiation, extending the study also to another specie of bird, the passeraceous zebrafinch. This observation aim to extend the knowledge on the histogenesis of barbs in feathers which is essential for further molecular studies on feather morphogenesis. The study is part of a large ultrastructural survey on feather morphogenesis aimed to clarify ans simplify the knowledged of all cell types involved in the formation of feathers.

MATERIAL AND METHOD

Chick embryos were used at progressive stages of development (Hamburger & Hamilton, 1951). Fertilized eggs were incubated at 37-40 °C and collected at 11 (n=3), 12 (n= 3), 13 (n=3), 14 (n=3), 15 (n=3), 16 (n=3), and 18 (n=3) days post-deposition. Within this time frame, the main stages in which feather morphogenesis occurs were obtained at Hamburger & Hamilton (HH) stages 36-40.

Developing feathers of the skin of dorsal, wing and ventral areas of the embryos, were collected and fixed for 5 h at 0-4°C in 2.5% glutaraldehyde and 4% paraformaldehyde in 0.1 M Phosphate buffer at pH 7.4. Tissues were rinsed in buffer, post-fixed in 2% osmium tetroxide for 90 min, dehydrated, and embedded in Durcupan resin. Developing feathers appeared as filaments of different lengths (HH stages 36-40). Other tissues were collected from embryos and from four juveniles (1 week old) of the passeraceous bird zebrafinch (Taniatopigigia castanotis) (see details in Alibardi & Sawyer). Fertilized eggs were collected at 11-12 days (n=3); 13-14 days (n=3), 14-15 days (n=2), 15-16 days (n=3). Embryonic feathers from the head, neck, wing, and low back were taken for the microscopic study. The embryos were sacrificed and the skin immediately fixed as reported below. In juveniles, after sacrificing the specimens, regenerating feathers from the wings, pectoral and dorsal skin were immediately fixed as above. Regenerating feathers appeared as 2-5 mm long cones (feather filaments) protruding from the skin.

Embryonic or juvenile feather filaments were sectioned at 1-3 |jm thickness in both cross, oblique and longitudinal planes using an ultramicrotome. Cross sections were collected from apical, intermediate, and basal parts of feather filaments at HH stages 36-40 in the chick, at 12-16 days post-deposition in zebrafinch, and in juvenile feathers of the zebrafinch. Some longitudinal, oblique and cross sections of 40-90 nm in thickness (thin sections) were also collected on copper grids for conventional ultrastructural examination. Thin sections were stained in uranyl acetate and lead citrate, and were observed under a CM-100 Philips electron microscope operating at 60-80 kV.

RESULTS

Histology of barb ridges. Unless specifically indicated the morfogénesis of feathers was similar in the two species. Feather filaments were surrounded by a multilayered sheath at stages 36-38 in the chick or 13-14 days post-deposition in the zebrafinch (Fig. 1 A). A similar histogenetic process was also noted in juvenile feathers of zebrafinch at 1 week post-hatching. Within these filaments long columns of cells were seen in longitudinal section which appeared as barb ridges in cross-section (Fig. 1A-C). A flat epithelium formed the

marginal plates bordering barb ridges (Fig. 1C, see ultrastructure later). The detailed examination of longitudinal sections of feather filaments showed the beginning of differentiation of barbs and barbules (Fig. ID). While barb medullary cells formed by piling up of cylindrical cells, barb cortical cells were seen as parallel fusiform cells forming a wall around medullary barb cells. More externally, fusiform barbule cells formed syncitial tubes reaching underneath the sheath. The innermost part of the sheath was made of pale, barb ridge vane cells. The central part of the feather filament contains mesenchymal cells and blood vessels.

At HH stages 39-40 in the chick large part of the feather filament (apical to more basal portions) was made of centrally located, keratinized barbs and by peripheral barbules (Figs. IE andF). In longitudinal sections, barb cells resemble tree-branches, with the thin barbules branching at regular intervals along the barb (ramus, Fig. IE). The branching of proximal (basal) barbule cells occured at opposite, right and left, sides on the barb. Barbules were made of several cells (5 or more) piled up to form narrow chains of barbule cells (see schematic drawing in Fig. 7). Barb medullary cells formed pale vacuolated centers surrounded by denserbarb cortical cells, the latter in continuity with barbule cells (Fig. 1 E, F). At late stages of feather morphogenesis (stage 40 in the chick and at 15-16 days post-deposition in zebrafinch) marginal plate cells and supportive cells present among barbule cells were largely disappeared. Large blood vessels were either degenerating or contained few blood cells.

Ultrastructure. An outer periderm and 3-5 layers of sheath cells were seen enwrapping the feather filaments at HH stages 37-38 in the chick and at 12-13 days in zebrafinch or in 1 week hatchlings. Pale barb ridge vane cells were interposed between the sheath externally and cells of barb ridges internally (Fig. 2). Cross sections of feather filaments in both species showed that barb ridges were made of a peripheral layer of cylindrical cells (presumptive marginal plates) and an inner axial plate made of irregularly aggregated cells (presumptive barb cells). The latter were in continuity with two external areas of more or less piled cells (alar plates, the presumptive barbule cells) separated by a central region of smaller cells (presumptive axial plate). Barb ridges were in contact with the external 4-5 cell-layered sheath by the interposition of paler cells with circular orientation, the barb vane ridge cells (Fig. 2).

At this stage of cytodifferentiation most cells of the barb ridge contained evenly distributed ribosomes, no endoplasmic reticulum and the nucleus was relatively euchromatic. However, barb and barbule cells often contained glycogen granules evenly distributed or clumped in variably-sized massess (Fig. 3). Vesicles of small size (0.1-0.4 |jm) were more or less evenly seen in the cytoplasm. The vesicles appeared to derive from the enlargment of the endoplasmic reticulum or from swelling and disappearing of cristae in mitochondria.

At later stages, 38-39 in the chick, 14-15 days and 1 week post-hatching in the zebrafinch, the vesicles in barb medullary cells increased and tended to fuse into large vesicles (Fig. 4). These vesicles appeared to contain a lipid-like material with very low density and their initial membrane appeared partially or largely interrupted (Fig. 3 B). Large lipid vesicles were more relevant in barb medullary cells of the zebrafinch, especially in feathers of 1 week post-hatchlings.

In the chick at HH stages 39-40 some amorphous material was still present within the large vesicles while keratin bundles accumulated along the cell periphery and joined the plasma membrane (Fig. 5 A). While the plasma membrane among barb cells showed small area of fusion or tight junctions at HH stages 37-38, large part of the membrane appered lost in the following stages, especially in rami of apical areas of feather filaments. At HH stages 39-40 some modified junctions were seen among barb cells, and they were composed by two dense peripheral lines and a thicker dense midline (Fig. 5 C). In the zebrafinch at 15-16 days post-deposition or in rami of feathers of 1 week post-hatchingls, most barb medullary cells were filled with lipids or partially empty (Fig. 6). While barb cortical cells were filled with keratin bundles, only the peripheral cytoplasm of barb medullary cells was keratinized forming trabeculae containing some lipids, occaional granular material (perhaps glycogen remnants), or empty cavities (Fig. 6). The lipidization of barb medullary cells was contemporary to that of interbarbule supportive cells at late stages of differentiation so that barbules became eventually independed and joined to the medullated ramus as indicated in Fig. 7.

DISCUSSION

The present ultrastructural observations on the phases of differentiation of cells of the ramus confirms and extends the previous, scanty descriptions (Matulionis; Alibardi, 2005a). Cells of the innermost ramus area of barb ridges becomes hypertrophic and rich in glycogen, either free or clumped in more or less extensive mass: this is the first cytological sign of barb medullary cell differentiation. The following morphological change in barb medullary cells is the formation of vesicles in their cytoplasm and numerous glycogen particles or clumps of glycogen remain attached to their membrane. The vesicles derive from both enlargment of the endoplasmic reticulum but others also from mithochondria swelling that merge with other vesicles to produce the larger lipid vesicles or droplets. It is likely that, at least part of the lipidie material that forms the droplets, can accumulate inside the degenerating mitochondria, as it occurs in other differentiating or in degenerating cells (La Via & Hill, 1975), including those of supportive cells of the feathers (Alibardi, 2005a,b). The degeneration of this organelles suggests that a process of anossia triggers the initial stages of degeneration of barb medullary cells that are relatively isolated from blood vessels (Matulionis). The rapid degeneration of these cells impede that they accumulate large amount of beta-keratin as in barb cortical and barbule cells.

The accumulation of lipids in barb medullary cells is different from that of adipocytes since in the former cells lipids form while both mitochondria and ergastoplasm disappear indicating a degenerative process (in adipocytes instead mitochondria and ergastoplasm remain active). It is likely that vesicles and the derived lipid droplets are derived from lipids of degenerating membrane of mitochondria, ergastoplasm and other cell organelled during cell degeneration. Also glycogen disappears or is incorporated into the degenerating vesicles and probably the degraded glucose is utilized to produce lipids.

In barb medullary cells the synthesis of feather keratin is limited in comparison that of both barb cortical and barbule cells (Filshie & Rogers, 1962; Matulionis; Kemp et al, 1974; Bowers & Brumbaugh, 1978; Alibardi, 2005a,b; Alibardi & Sawyer). It remains to be identified the mechanism that address the metabolic utilization of glycogen to produce lipids instead of keratin. As a result, in the small region of the ramus area of barb ridges the central cells change their differentiative fate from a keratin-producing cell to a lipid producing cell.

It is known that in the epidermis of reptiles and birds only differentiating alpha-keratin cells can accumulate large amount of lipids while cells synthezing beta-keratin accumulate little lipids. The accumulation of lipid material in beta-cells has been described only in some layers of scale of some lizard species where cells become glandular (Maderson & Alibardi, 2000). The mechanism of change of their differentiative fate isunknown.

Aside the descriptive aspects the present observations emphasise that the lipidogenic potential of avian keratinoeyets is present not only in avian alpha-keratinocyets (sebokeratinocytes) but also in beta-keratinocytes of feathers. The mechanism respsonsible for the loss of the capability to synthezise feather keratin in the central mass of barb cells remains to be identified. As a result, the control of lipidogenesis in beta-cells of the medulla of a ramus may reverts to that of alpha-cells in order to create empty spaces that ensure the lightness of feathers coupled to the resistence of the wall of rami. Furthermore the empty spaces in rami contribute to the physical process of light interference and scattering responsible for the physical colouration of feathers (Lucas & Stettenheim; Spearman & Hardy, 1985).

ACKNOWLEDGMENTS

The study was partially supported by a University of Bologna 60% grant and largely by self-support. Dr. Mattia Toni skilfully helped in the computer drawing of Fig. 1 using Corel Draw Program.

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Correspondence to: Dr. Lorenzo Alibardi
Dipartimento di Biología Evoluzionistica Sperimentale, University of Bologna. ViaSelmi3, 40126. Bologna.
ITALY

Received: 19-10-2006, Accepted: 15-01-2007